Magnetic beads have gained increasing use as a convenient separation technique for many forms of cell, nucleic acid and protein isolations and analyses. In particular, the manufacturers of clinical immunoassays utilize magnetic beads both as a solid support for antibodies specifically targeted to analytes of clinical importance, and as the separation means to isolate and detect those bound analytes. But use of magnetic beads imposes a paradox. The magnetic beads must have sufficient size - normally on the order of several microns in diameter - in order to be separated in an easily achievable magnetic field. But magnetic beads of this size diffuse only very slowly, and present limited surface area for antibody binding compared to their volume. Thus the size of the current magnetic beads limits the speed and sensitivity that can be achieved in clinical assays. Through advances in template-directed polymer synthesis and nanotechnology, a new class of magnetic nanoparticles can be made. These magnetic nanoparticles, bearing stimuli-responsive polymers, can change from a monodispersed small diameter particle of roughly 20 nanometers diameter to a macro- aggregate of microns diameter in response to an environmental stimulus like a temperature or pH change. By using these advanced nanomaterials, assays can be developed in which the high surface area to volume ratio and the small size / high diffusion of the magnetic nanoparticles provides for higher sensitivity and faster binding reactions compared to current magnetic bead reagents. And then a discrete pH or temperature stimulus can cause these nanoparticle reagents to rapidly co-aggregate into a macro-complex of micron dimensions which can be separated by a magnetic field as easily and quickly as currently used magnetic beads. This project will develop, optimize, and begin scale-up of these unique assay reagents, and will then demonstrate their value in a model p24 (HIV) protein immunoassay. Phase II of this project will also demonstrate the ability to multiplex various types of HIV tests using these reagents, and demonstrate the potential of removing serum interferences for certain important clinical analytes. The HIV target assay of this project is only a model. These materials present the promise of faster, more sensitive clinical immunoassays for important biomarkers of cardiac disease, cancer, endocrine disorders and other infectious diseases. The multiplex capabilities of these reagents and the potential for removal of serum interferences will be broadly applicable to other clinically important analytes.